Bacterial lysates improve the protective antibody response against respiratory viruses through Toll-like receptor 4.

Respiratory viruses cause significant morbidity and mortality in infants and young children worldwide. Current strategies to modulate the immune system and prevent or treat respiratory viral infections in this age group have shown limited success. Here, we demonstrate that a lysate derived from Gram-positive and Gram-negative organisms positively modulates protective antibody responses against both respiratory syncytial virus (RSV) and influenza virus in murine models of infection. Interestingly, despite the complex mixture of Toll-like receptor (TLR) agonists present in the bacterial lysate, the modulatory effects were mostly dependent on TLR4 signaling. Our results indicate that the use of simple formulations of TLR-agonists can significantly improve the immune response against critical pediatric respiratory pathogens.

T helper type 2 bias and type 17 suppression in primary dengue virus infection in infants and young children.

BACKGROUND: The immune response to dengue virus (DENV) primary infection in infants and young children is not well characterized. In Northern Argentina, >90% of the population was DENV-naïve before the 2009 outbreak, allowing evaluation of age-dependent primary responses to infection. METHODS: We conducted a comparative study of the immune response to DENV in 27 infected infants, young children and their mothers. Lymphocyte T helper (Th) 1, Th2, Th17 and inflammatory responses were assayed in blood during the 2009 DENV-1 epidemic. RESULTS: The immune response to DENV-1 was significantly biased to Th2 in infected infants and young children, compared to infants with other febrile illnesses (for IL-4 p < 0.001) and to their infected mothers (for IL-4 p < 0.01). In addition, IL-17 suppression was observed in the memory response to DENV-1 in infected infants (p < 0.01 vs placebo). CONCLUSION: Age-related differences in the primary response to DENV, characterized by an immature Th2 polarization and Th17 suppression in infants, should be studied further in order to expand our understanding of the mechanism of dengue pathogenesis.

Macronutrients during pregnancy and life-threatening respiratory syncytial virus infections in children.

RATIONALE: Respiratory syncytial virus (RSV) is an important cause of hospitalization and death in infants worldwide. Most RSV deaths occur in developing countries, where burden and risk factors for life-threatening illness are unclear. OBJECTIVES: We defined the burden of life-threatening (O(2) saturation [O(2) sat] ≤ 87%) and fatal RSV infection, and characterized risk factors for life-threatening disease in hospitalized children. Special emphasis was placed on studying the impact of dietary habits during pregnancy. We hypothesized that dietary preferences, differing from those of our remote ancestors, would negatively impact children's pulmonary health. For instance, a diet rich in carbohydrates is a signature of recent millennia and typical of low-income populations, heavily burdened by life-threatening RSV disease. METHODS: Prospective study in a catchment population of 56,560 children under 2 years of age during the RSV season in Argentina. All children with respiratory signs and O(2) sat less than 93% on admission were included. MEASUREMENTS AND MAIN RESULTS: Among 1,293 children with respiratory infections, 797(61.6%) were infected with RSV: 106 of these had life-threatening disease; 1.9 per 1,000 children (95% confidence interval [CI], 1.5-2.2/1,000) under 24 months. A total of 22 hospitalized children died (9 RSV(+)), 26 died at home due to acute respiratory infection (14 attributed to RSV); all were under 12 months old. The annual attributable mortality rate for RSV was 0.7 per 1,000 infants (95% CI, 0.4-1.1/1,000). Life-threatening disease was dose-dependently associated with carbohydrate ingestion during pregnancy (adjusted odds ratio from 3.29 [95% CI, 1.15-9.44] to 7.36 [95% CI, 2.41-22.5] versus the lowest quartile). CONCLUSIONS: Life-threatening and fatal RSV infections are a heavy burden on infants in the developing world. Diets rich in carbohydrates during pregnancy are associated with these severe outcomes.

A mechanistic role for type III IFN-λ1 in asthma exacerbations mediated by human rhinoviruses.

RATIONALE: Human rhinoviruses (HRV) are the leading cause of upper respiratory infections and have been postulated to trigger asthma exacerbations. However, whether HRV are detected during crises because upper respiratory infections often accompany asthma attacks, or because they specifically elicit exacerbations, is unclear. Moreover, although several hypotheses have been advanced to explain virus-induced exacerbations, their mechanism remains unclear. OBJECTIVES: To determine the role of HRV in pediatric asthma exacerbations and the mechanisms mediating wheezing. METHODS: We prospectively studied 409 children with asthma presenting with upper respiratory infection in the presence or absence of wheezing. Candidate viral and immune mediators of illness were compared among children with asthma with different degrees of severity of acute asthma. MEASUREMENTS AND MAIN RESULTS: HRV infections specifically associated with asthma exacerbations, even after adjusting for relevant demographic and clinical variables defined a priori (odds ratio, 1.90; 95% confidence interval, 1.21-2.99; P = 0.005). No difference in virus titers, HRV species, and inflammatory or allergic molecules was observed between wheezing and nonwheezing children infected with HRV. Type III IFN-λ(1) levels were higher in wheezing children infected with HRV compared with nonwheezing (P < 0.001) and increased with worsening symptoms (P < 0.001). Moreover, after adjusting for IFN-λ(1), children with asthma infected with HRV were no longer more likely to wheeze than those who were HRV-negative (odds ratio, 1.18; 95% confidence interval, 0.57-2.46; P = 0.66). CONCLUSIONS: Our findings suggest that HRV infections in children with asthma are specifically associated with acute wheezing, and that type III IFN-λ(1) responses mediate exacerbations caused by HRV. Modulation of IFN- λ(1) should be studied as a therapeutic target for exacerbations caused by HRV.

Delivery with polycations extends the immunostimulant Ribomunyl into a potent antiviral Toll-like receptor 7/8 agonist.

BACKGROUND: Upper respiratory tract infection is a frequent cause of morbidity worldwide. Although the course of infection is usually mild, it is responsible for enormous social and economic costs. Immunostimulation with bacterial extracts consisting of ribosomal RNA and proteoglycans, such as Ribomunyl, was introduced into the clinic in the 1980s as a new treatment concept, but did not achieve widespread application. Ribomunyl has been proposed to activate innate immunity, but the contribution of its RNA content as well as its antiviral potential has not been studied. METHODS: Peripheral blood mononuclear cells from healthy donors and immune cells from adenoids were incubated with Ribomunyl either by itself or formulated in a complex with cationic polypeptides such as poly-l-arginine or protamine, and induction of cytokines was quantified by ELISA. RESULTS: Ribomunyl in complex with either poly-l-arginine or protamine, but not on its own, was able to strongly induce interferon-α (P<0.01) and interleukin-12 (P<0.01) in peripheral blood mononuclear cells, whereas induced tumour necrosis factor-α and interleukin-6 levels were independent of the formulation. Comparable results were obtained in immune cells from adenoids, suggesting efficacy also in virus-affected tissue. Cell sorting, RNase digests and selective receptor expression show that the RNA in Ribomunyl acts as an agonist of Toll-like receptor (TLR)7 and TLR8. CONCLUSIONS: Ribomunyl is, in principle, able to potently induce antiviral interferon-α and interleukin-12 via TLR7 and TLR8, respectively, but only when formulated in a complex with cationic polypeptides.

Immunogenic cell death of human ovarian cancer cells induced by cytosolic poly(I:C) leads to myeloid cell maturation and activates NK cells.

Owing to high rates of tumor relapse, ovarian cancer remains a fatal disease for which new therapeutic approaches are urgently needed. Accumulating evidence indicates that immune stimulation may delay or even prevent disease recurrence in ovarian cancer. In order to elicit proinflammatory signals that induce or amplify antitumor immune reactivity, we mimicked viral infection in ascites-derived ovarian cancer cells. By transfection or electroporation we targeted the synthetic double-stranded RNA poly(I:C) intracellularly in order to activate melanoma differentiation-associated gene-5 (MDA-5), a sensor of viral RNA in the cytosol of somatic cells. Cancer cells reacted with enhanced expression of HLA-class I, release of CXCL10, IL-6, and type I IFN as well as tumor cell apoptosis. Monocytes and monocyte-derived DCs (MoDCs) engulfed MDA-5-activated cancer cells, and subsequently upregulated HLA-class I/II and costimulatory molecules, and secreted CXCL10 and IFN-α. Further, this proinflammatory milieu promoted cytolytic activity and IFN-γ secretion of NK cells. Thus, our data suggest that the engagement of MDA-5 in a whole tumor cell vaccine is a promising approach for the immunotherapy of ovarian cancer.

Higher activation of TLR9 in plasmacytoid dendritic cells by microbial DNA compared with self-DNA based on CpG-specific recognition of phosphodiester DNA.

TLR9 detects DNA in endolysosomal compartments of human B cells and PDC. Recently, the concept of the CpG motif specificity of TLR9-mediated detection, specifically of natural phosphodiester DNA, has been challenged. Unlike in human B cells, CpG specificity of natural phosphodiester DNA recognition in human PDC has not been analyzed in the literature. Here, we found that the induction of IFN-alpha and TNF-alpha in human PDC by phosphodiester ODNs containing one or two CG dinucleotides was reduced to a lower level when the CG dinucleotides were methylated and was abolished if the CGs were switched to GCs. Consistent with a high frequency of unmethylated CG dinucleotides, bacterial DNA induced high levels of IFN-alpha in PDC; IFN-alpha was reduced but not abolished upon methylation of bacterial DNA. Mammalian DNA containing low numbers of CG dinucleotides, which are frequently methylated, induced IFN-alpha in PDC consistently but on a much lower level than bacterial DNA. For activation of PDC, phosphodiester ODNs and genomic DNA strictly required complexation with cationic molecules such as the keratinocyte-derived antimicrobial peptide LL37 or a scrambled derivative. In conclusion, we demonstrate that self-DNA complexed to cationic molecules activate PDC and thus, indeed, may function as DAMPs; nevertheless, the preference of PDC for CpG containing DNA provides the basis for the discrimination of microbial from self-DNA even if DNA is presented in the condensed form of a complex.

Recognition of 5' triphosphate by RIG-I helicase requires short blunt double-stranded RNA as contained in panhandle of negative-strand virus.

Antiviral immunity is triggered by immunorecognition of viral nucleic acids. The cytosolic helicase RIG-I is a key sensor of viral infections and is activated by RNA containing a triphosphate at the 5' end. The exact structure of RNA activating RIG-I remains controversial. Here, we established a chemical approach for 5' triphosphate oligoribonucleotide synthesis and found that synthetic single-stranded 5' triphosphate oligoribonucleotides were unable to bind and activate RIG-I. Conversely, the addition of the synthetic complementary strand resulted in optimal binding and activation of RIG-I. Short double-strand conformation with base pairing of the nucleoside carrying the 5' triphosphate was required. RIG-I activation was impaired by a 3' overhang at the 5' triphosphate end. These results define the structure of RNA for full RIG-I activation and explain how RIG-I detects negative-strand RNA viruses that lack long double-stranded RNA but do contain blunt short double-stranded 5' triphosphate RNA in the panhandle region of their single-stranded genome.

Selective and direct activation of human neutrophils but not eosinophils by Toll-like receptor 8.

BACKGROUND: Granulocytes represent the largest fraction of immune cells in peripheral blood and are directly exposed to circulating Toll-like receptor (TLR) ligands. Although highly relevant for TLR-based therapies, because of the technical challenge, activation of the granulocyte subsets of neutrophils and eosinophils by TLR ligands is less well studied than activation of other immune cell subsets. OBJECTIVE: The aim of this work was to study direct versus indirect neutrophil and eosinophil activation by TLR7 and TLR8 ligands. METHODS: We used a new whole-blood assay, single cell-based cytokine detection, and highly purified primary human neutrophils and eosinophils to separate direct and indirect effects on these blood cell subsets. RESULTS: We found indirect but not direct activation of neutrophils but not eosinophils in whole blood by using unmodified immunostimulatory RNA (isRNA; TLR7/8 ligand). In contrast, direct activation and stimulation of the respiratory burst and degranulation was seen with nuclease-stable isRNA and with the small-molecule TLR8 agonist 3M002 but not 3M001 (TLR7). Neutrophils expressed TLR8 but none of the other 2 RNA-detecting TLRs (TLR3 and TLR7). CONCLUSIONS: Together, these results demonstrate that neutrophils are directly and fully activated through TLR8 but not TLR7. Furthermore, the results predict that the clinical utility of small-molecule TLR8 ligands or nuclease-stable RNA ligands for TLR8 might be limited because of neutrophil-mediated toxicity and that no such limitation applies for unmodified isRNA, which is known to induce desired T(H)1 activities in other immune cell subsets.

Approaching the RNA ligand for RIG-I?

Innate and antigen-specific antiviral immunity are triggered by immunorecognition of viral nucleic acids. The helicase retinoic acid-inducible gene I (RIG-I) (also known as DDX58) is the key sensor of negative strand RNA viruses in the cytosol of cells. RNA containing a triphosphate at the 5'-end was shown to activate RIG-I, but the exact structure of RNA supporting 5'-triphosphate recognition, the requirement of a 5'-triphosphate group, as well as the existence of RNA structures detected by RIG-I in the absence of 5'-triphosphate remain controversial. Here, we revisit the literature on RIG-I and RIG-I ligands. The literature proposes at least six different RIG-I ligands: (i) single strand with a 5'-triphosphate, (ii) double-stranded RNA with a 5'-triphosphate, (iii) 5'-triphosphate single-stranded RNA with A- and U-rich 3'-sequences, (iv) double-stranded RNA of intermediate length (>300 and <2000 bp) without 5'-triphosphate, (v) blunt-end short double-stranded RNA (23-30 bp) without 5'-triphosphate, and (vi) short double-stranded RNA (23-30 bp) with 5'-monophosphate. RIG-I thus seems promiscuous for a variety of different RNA molecules, very similar to the Toll-like receptors, of which 10 family members are sufficient for the safe detection of the microbial cosmos. In the light of these outstanding publications, it seems an unlikely possibility that there is a fundamental shortcoming in the design of all studies. Looking closely, the only issue that comes to mind is the in vitro transcription technique used by all investigators without confirming the identity of RNA products. This technique, together with the different biological systems used, the lack of dose responses and of proper comparison of different published ligands and controls leave us with more questions than answers as to what the exact RIG-I ligand is, if in fact it exists.

Accessing the therapeutic potential of immunostimulatory nucleic acids.

Short synthetic CpG oligodesoxynucleotides (CpG ODN) that activate plasmacytoid dendritic cells (PDC) and B-cells via the endosomal Toll-like receptor (TLR) 9 are the prototypical immunostimulatory nucleic acids (NAs), and their therapeutic potential is currently evaluated in numerous clinical trials. In recent years, NA recognition has emerged as a general means of virus detection by the innate immune system that involves at least seven receptors located in either the endosome or the cytosol. Endosomal TLRs are expressed selectively, and predominantly by immune cell types. By contrast, the expression of cytosolic NA receptors is ubiquitous, significantly widening the range of cell types that can be stimulated therapeutically by NAs to include even tumor cells. Here we discuss unique properties of each of these receptors, and argue that an understanding of the molecular basis of receptor-ligand interactions, and the development of chemically defined, selective ligands is required in order to fully realize the promise that NA immunetherapeutics hold.